Lung cancer is the leading cause of cancer death in American men and women, and only 13 percent of people who develop lung cancer survive 5 years. Supportive care for non-small cell lung cancer, which is the most common form, yields median survival rates of just 4 months. New drug combinations improve survival only to 8-10 months. The development of new treatment modalities is therefore of great importance. The research detailed in this proposal aims to develop novel, highly effective, but safe gene therapy approaches to treat lung cancer. The approach will be based on modifying adenoviral genes to target viral replication and cell killing to lung cancer cells. Cell cycle regulating genes such as p53 and Rb are inactivated both in cancer cells and in adenovirus infected cells. Deletion of the adenoviral genes that alter control of the cell cycle may therefore target viral replication to cancer cells. An Elb-55kD deleted virus has been introduced to target p53 mutant cancer cells, however several reports have questioned this approach. In this proposal, it will be determined if an adenovirus with a modified Ela gene that is unable to inactivate Rb and/or p300, will target viral replication to cancer cells. To further target viral replication to lung cancer cells, transcription of the modified Ela gene will be restricted to lung cells by using lung specific promoters. The focus of the second aim will be improving the oncolytic activity of a replicating adenoviral vector. The adenoviral E1b-19kD protein is a potent inhibitor of apoptosis, and an adenovirus with this gene deleted, more efficiently kills and spreads through a monolayer of tumor cells. The effects of the E1b-19kD deletion will therefore now be evaluated in a mouse model. In addition, the effects of combining viral infection with cytotoxic agents will be evaluated. In the third aim, the oncolytic activity and specificity of an adenoviral vector, that combines transcriptional targeting of the modified Ela gene with an E1b-19kD gene deletion will be evaluated. Safety aspects are difficult to evaluate in a mouse model as human adenoviruses do not replicate in mouse cells. In the fourth aim, the safety of these adenoviral constructs will be evaluating by measurement of viral replication, cell killing and induction of apoptosis in freshly isolated lung tumor cells.